POS0239 JAK-INHIBITORS SUPPRESS IFN-DEPENDENT ANTIGEN PRESENTATION IN CDC42HICD14+ CELLS
Authors: E. Malmhäll-Bah, K. M. Andersson, S. Töyrä Silfverswärd, M. Erlandsson, R. Pullertis, M. I. Bokarewa
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> We have recently shown that Rho GTPase activation in macrophages results in arthritis development in mice. These macrophages show a tight interaction with T cells leading to suppression of cell homing and regulate migration of thymic T regulatory cells to periphery contributing arthritis development<sup>[1]</sup>. <h3>Objectives</h3> To translate findings in experimental arthritis to human RA, we explore the phenotype of <i>CDC42</i><sup><i>hi</i></sup>CD14<sup>+</sup> cells in blood and synovial tissue of RA patients and investigate how anti-rheumatic treatment intervene with the Rho GTPase-dependent molecular mechanisms of arthritis. <h3>Methods</h3> We used transcriptome (RNAseq) of CD14<sup>+</sup> and CD4<sup>+</sup> cells of 77 active RA patients naïve to TNF-inhibitors (TNFi) and of 59 inactive RA patients treated with methotrexate (MTX, n=18), TNFi (n=10) and JAK-inhibitors (JAKi, n=24), or having no DMARD treatment (n=7). In both cohorts, patients were stratified by mean expression of <i>CDC42</i> in CD14<sup>+</sup> cells to mimic activation of Rho GTPases. Differentially expressed genes (DEG) were identified by DESeq2 (nominal p&lt;0.05). Transcriptomics of CD14<sup>+</sup> and CD4<sup>+</sup> cells were analyzed and translated into clinical correlates of inflammation and disease activity by DAS28. Additionally, we investigated a cluster of <i>CDC42</i><sup><i>hi</i></sup> macrophages in synovial tissue (STM) using single cell RNAseq in 25 patients<sup>[2]</sup>. <h3>Results</h3> In two independent sets, <i>CDC42</i><sup><i>hi</i></sup>CD14<sup>+</sup> cells were characterized by enrichment of DEG operating in the oxidative phosphorylation, proteasome activity and RNA transcription. Many DEG were under transcriptional control of NFE2L1, known to supervise those processes. DEG with strong correlation to <i>CDC42</i> defined the metabolic signature (MetSig) of <i>CDC42</i><sup><i>hi</i></sup>CD14<sup>+</sup> cells, which summarized the expression of <i>ATP5BP</i>, <i>COX7A2</i>, <i>PSMB6</i>, <i>PSME3, GTF3C6,</i> and <i>GTF2E2</i>. High MetSig recognized CD14<sup>+</sup> cells enriched with MHC transcripts, components in peptide loading and expression of proteasome subunits. MetSig<sup>hi</sup>CD14<sup>+</sup> cells had high chemokine and cytokine production and migratory phenotype. Analyzing STM, we identified <i>CDC42</i><sup><i>hi</i></sup>STM with high MetSig expressing complete set of immunoproteasome genes, high levels of MHC receptors and peptide loading proteins. Additionally, this STM cluster was affected by IFN-γ. We experimentally confirmed that immunoproteasome expression in CD14<sup>+</sup> cells was dependent on IFN-γ and not hypoxia, common at inflammation. Examining effect of antirheumatic treatment, we found <i>CDC42</i><sup><i>hi</i></sup>CD14<sup>+</sup> cells were infrequent (<i>OR</i>= 11.9, [3.4, 49.7], <i>p</i>&lt;0.0001) and the MetSig reduced (<i>p</i>=0.0004) in patients treated with JAKi compared to other DMARDs. Concurrently, CD14<sup>+</sup> cells of those patients had low production of TNF-α, IL-6, IL-1β, CXCL8 and IL-10 (all, <i>p</i>&lt;0.05). mRNA of immunoproteasome subunits, peptide loading proteins and MHC-II (including <i>HLA-DRB1</i>) were downregulated. Analysis of CD4<sup>+</sup> cells of JAKi treated patients showed upregulation of TCR components and high expression of Rho GTPases. This indicated a disruption of CD14<sup>+</sup>/CD4<sup>+</sup> interaction in the immunological synapse. To investigate a direct effect of JAKi on the MetSig genes and immunoproteasome, we treated CD14<sup>+</sup> cell cultures with tofacitinib. JAKi upregulated <i>NFE2L1</i> and decreased expression of the immunoproteasome subunits, but expression of MetSig remained unchanged. Lastly, we investigated connection between the MetSig and RA disease activity (by DAS28) in a regression model. The model identified patients, which had a strong dependence between these parameters. At bottom of the model were patients with low DAS28 and MetSig, mostly treated with JAKi. Consequently, patients at the upper part may represent suitable candidates for JAKi-treatment. <h3>Conclusion</h3> This study demonstrates that CDC42-related metabolic signature identifies antigen-presenting CD14<sup>+</sup> cells that migrate to joints to coordinate autoimmunity. JAKi suppress the antigen presenting capacity of CD14<sup>+</sup> cells. The MetSig in CD14<sup>+</sup> cells may identify candidate patients for JAKi treatment. <h3>References</h3> [1]Malmhäll-Bah, et al. J Autoimmun. <b>130</b>, 102843 2022. [2]Alivernini, et al. Nat Med. <b>26</b>(8), 1295-1306 2020. <h3>Acknowledgements</h3> We thank the research nurses Anneli Lund and Marie-Louise Andersson at Rheumatology Clinic, Sahlgrenska University Hospital, Gothenburg, for blood sampling. We thank all RA patients who participated. We appreciate support of Aridaman Pandit and Weiyang Tao at Center for Translational Immunology, University Medical Center Utrecht, NL, sharing data of the BiOCURA cohort. We are thankful to Prof. Marcela Pekna at Clinical Neuroscience, University of Gothenburg, assisting with hypoxia experiments. <h3>Disclosure of Interests</h3> None Declared.